SARS-CoV-2 Omicron potently neutralized by a novel antibody with unique Spike binding properties
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Abstract
The SARS-CoV-2 Omicron variant exhibits very high levels of transmission, pronounced resistance to authorized therapeutic human monoclonal antibodies and reduced sensitivity to vaccine-induced immunity. Here we describe P2G3, a human monoclonal antibody (mAb) isolated from a previously infected and vaccinated donor, which displays picomolar-range neutralizing activity against Omicron BA.1, BA.1.1, BA.2 and all other current variants, and is thus markedly more potent than all authorized or clinically advanced anti-SARS-CoV-2 mAbs. Structural characterization of P2G3 Fab in complex with the Omicron Spike demonstrates unique binding properties to both down and up spike trimer conformations at an epitope that partially overlaps with the receptor-binding domain (RBD), yet is distinct from those bound by all other characterized mAbs. This distinct epitope and angle of attack allows P2G3 to overcome all the Omicron mutations abolishing or impairing neutralization by other anti-SARS-COV-2 mAbs, and P2G3 accordingly confers complete prophylactic protection in the SARS-CoV-2 Omicron monkey challenge model. Finally, although we could isolate in vitro SARS-CoV2 mutants escaping neutralization by P2G3 or by P5C3, a previously described broadly active Class 1 mAb, we found these viruses to be lowly infectious and their key mutations extremely rare in the wild, and we could demonstrate that P2G3/P5C3 efficiently cross-neutralized one another’s escapees. We conclude that this combination of mAbs has great prospects in both the prophylactic and therapeutic settings to protect from Omicron and other VOCs.
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SciScore for 10.1101/2022.03.18.484873: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Study design and use of subject samples were approved by the Institutional Review Board of the Lausanne University Hospital and the ‘Commission d’éthique du Canton de Vaud’ (CER-VD). Sex as a biological variable Four female cynomolgus macaques aged 3–6 years were randomly assigned between the control and treated groups to evaluate the efficacy of P2G3 LS in the prophylaxis challenge study. Randomization Four female cynomolgus macaques aged 3–6 years were randomly assigned between the control and treated groups to evaluate the efficacy of P2G3 LS in the prophylaxis challenge study. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies SciScore for 10.1101/2022.03.18.484873: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Study design and use of subject samples were approved by the Institutional Review Board of the Lausanne University Hospital and the ‘Commission d’éthique du Canton de Vaud’ (CER-VD). Sex as a biological variable Four female cynomolgus macaques aged 3–6 years were randomly assigned between the control and treated groups to evaluate the efficacy of P2G3 LS in the prophylaxis challenge study. Randomization Four female cynomolgus macaques aged 3–6 years were randomly assigned between the control and treated groups to evaluate the efficacy of P2G3 LS in the prophylaxis challenge study. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Spike and RBD tetramers were prepared fresh before use and formed by combining biotinylated proteins with PE-conjugated Streptavidin (BD Biosciences) at a molar ratio of 4:1. Binding and ACE2 blocking studies with SARS-CoV-2 Spike: Luminex beads used for the serological and purified antibody binding assays were prepared by covalent coupling of SARS-CoV-2 proteins with MagPlex beads using the manufacturer’s protocol with a Bio-Plex Amine Coupling Kit (Bio-Rad, France). ACE2suggested: NoneBiotinylated P5C3, P2G3, REGN10933, REGN10987, AZD8895, AZD1061, ADG-2 or S309 antibodies (prepared as described above) were added to each well at 1 μg/ml followed by a further 20-minute incubation. AZD8895suggested: NoneS309suggested: NoneFreshly isolated cells were stained with the cocktail of fluorescent conjugated antibodies containing anti-CD19 APC-Cy7, anti-CD3-BV510, anti-IgM-FITC, anti-IgD PE-CF594, anti-CD27-APC, anti-CD38-V450 (BD Biosciences) along with the pre-complexed Beta variant Spike tetramer (2 μg in 100μl) coupled to PE-streptavidin (BD Biosciences). anti-CD19 APC-Cy7, anti-CD3-BV510, anti-IgM-FITC, anti-IgD PE-CF594suggested: Noneanti-IgDsuggested: Noneanti-CD27-APCsuggested: (Sigma-Aldrich Cat# SAB4700132, RRID:AB_10896618)anti-CD38-V450suggested: NoneThe treated group (n = 2 [MF1 and MF2]) received one dose at 10 mg/kg of P2G3 LS human IgG1 monoclonal antibody delivered by intravenous injection three day prior to challenge, while control animals (n = 2 in parallel [MF3 and MF4] and n=2 historical [MF5 and MF6]) received no treatment. human IgG1suggested: NoneMF4suggested: NoneIn the ADCC assay, CEM-NKR-Spike-Luc cells were incubations with anti-Spike antibody at 0.3 μg/ml, isotype control antibodies at 0.3 μg/ml or an anti-HLA class I (MHC) positive control antibody (Invivogen) at 0.005 μg/ml. anti-Spikesuggested: NoneI (MHC) positive control antibody (Invivogen) at 0.005suggested: NoneTo monitor cell killing, co-cultured cells were washed and stained with fluorescent conjugated antibodies, CD56-AF488 (BD Biosciences,), CD16-FITC (BD Biosciences), Aqua Live/Dead cell stain (Invitrogen), CD4-PECF594 (BD Biosciences) and Annexin V-APC (BD Biosciences), and then analysed using a FACS LSR II cytometer instrument. Annexin V-APCsuggested: NonePositive control anti-MHC (anti-HLA class I) antibody gave strong antibody dependent cell killing of 60-80% and anti-Spike antibodies gave intermediate responses. anti-MHCsuggested: Noneanti-HLA class Isuggested: NoneExperimental Models: Cell Lines Sentences Resources Viral stocks were prepared in EPISERF on VeroE6 or Calu-3 (for Omicron), aliquoted, frozen and titrated on VeroE6 cells. VeroE6suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)Rev and pUltra-Chili-Luc vectors (Addgene) into 293T cells in DMEM medium + 10% FCS using Fugene 6 (Promega) for pseudoviruses production. 293Tsuggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)All animals were then exposed to a total dose of 105 TCID50 of Omicron B.1.1.529 SARS-CoV-2 virus produced in Calu-3 cells (NIH/BEI reference: NR-56462) via the combination of intranasal and intratracheal routes (day 0) with sample collection and testing performed as previously described 36. Calu-3suggested: NoneU937 cells incubated with Spike beads in the absence of antibody generally showed <5% phagocytic activity while increased ADCP activity was observed with increasing concentration of anti-Spike antibody with a maximum of 100% of cells exhibiting fluorescence associated with Spike bead phagocytosis. U937suggested: NoneRecombinant DNA Sentences Resources The Omicron Spike ectodomain was amplified by PCR with primers (listed in Table 3) designed on consensus sequence from available Omicron sequences, and introduced by In-Fusion cloning into the nCoV-2P-F3CH2S plasmid, replacing the original wild-type Spike29. nCoV-2P-F3CH2Ssuggested: NonePseudoviruses were alternatively produced with the original 2019-nCoV (Cat #100976), Alpha / B.1.1.7 (Cat #101023) and Beta/B.1.351 (Cat #101024) pCAGGS-SARS2-Spike vectors obtained from NIBSC. pCAGGS-SARS2-Spikesuggested: NoneThese vectors were co-transfected with pMDL p.RRE, pRSV. pMDL p.RREsuggested: NonepRSVsuggested: RRID:Addgene_106453)Rev and pUltra-Chili-Luc vectors (Addgene) into 293T cells in DMEM medium + 10% FCS using Fugene 6 (Promega) for pseudoviruses production. pUltra-Chili-Lucsuggested: RRID:Addgene_48688)Software and Algorithms Sentences Resources To monitor cell killing, co-cultured cells were washed and stained with fluorescent conjugated antibodies, CD56-AF488 (BD Biosciences,), CD16-FITC (BD Biosciences), Aqua Live/Dead cell stain (Invitrogen), CD4-PECF594 (BD Biosciences) and Annexin V-APC (BD Biosciences), and then analysed using a FACS LSR II cytometer instrument. BD Biosciencessuggested: (BD Biosciences, RRID:SCR_013311)The soft mask volume were generated manually in UCSF Chimera and cryoSPARC 40. cryoSPARCsuggested: (cryoSPARC, RRID:SCR_016501)Cryo-electron microscopy model building: A model of a Spike trimer (PDB ID 7QO7) or AlphaFold2 (ColabFold implementation) models of the P5C3 and P2G3 Fabs were fit into the cryo-EM maps with UCSF Chimera These docked models were extended and rebuilt manually with refinement, using Coot and Phenix41,42. Cootsuggested: (Coot, RRID:SCR_014222)Buried surface area measurements and centroid measurements were calculated within ChimeraX. ChimeraXsuggested: (UCSF ChimeraX, RRID:SCR_015872)Analyses were performed in GraphPad Prism (GraphPad Software, Inc.) and Microsoft Excel. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Microsoft Excelsuggested: (Microsoft Excel, RRID:SCR_016137)Results from OddPub: Thank you for sharing your data.
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
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- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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